Characterization of ubiquitination of histones H2A and H3 and ubiquitin-proteasome pathway gene expression during erythroid terminal differentiation
Parmar, Abhishek A.
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Ubiquitin is a eukaryotic protein which is associated with a number of processes such as cell cycle regulation, DNA repair and protein turnover. The proteins meant for degradation are tagged by multiple units of ubiquitin via a process known as ubiquitination. The ubiquitin proteasome pathway is involved in removal of proteins during the process of erythroid terminal differentiation (ETD). Histone H2A and H3 are proteins involved in packaging of the DNA within the cell's nucleus. Previous studies suggested that ubiquitination of histones may play an important role in modification of chromatin during ETD. This thesis project focused on analyzing the ubiquitination status of H2A & H3 during ETD and studying the expression of genes involved in the ubiquitin proteasome pathway during ETD. The ubiquitination of histone H2A and H3 was studied by immunoprecipitating these proteins using protein G coated magnetic beads followed by western blot analysis using anti ubiquitin antibody. The exact ubiquitination pattern could not be determined since the immunoprecipitation did not work in the desired manner. Western blot analysis of total histones using anti-ubiquitin antibody showed the possible presence of mono-ubiquitinated histone H2A and di-ubiquitinated H3 but this could not be confirmed since the anti-histone H2A & anti histone H3 western blots could not be colocalized with the ubiquitinated bands on the gel . The gene expression studies were done using focused qPCR arrays which had 84 genes involved in the ubiquitin proteasome pathway arranged in individual wells of the arrays. A total of three replicates were performed and eight genes showed a change in their expressions. Six genes on the PCR arrays were found to be upregulated significantly which included Park2, Ube2b, Ube2h, Ube2e3, Uba3 & Trp 53. Two genes, BRCA1 & Ube2t were found to be down regulated significantly. The up regulated genes included two ubiquitin conjugating enzyme Ube2b (HR6B) & Ube2h (E2-20K) independently determined to be upregulated by northern blot analysis from previous studies. Only four genes, out of the various ubiquitin conjugating enzymes present on the array showed a change in their expression. This showed that the expression of genes associated with the ubiquitin proteasome pathway during ETD, was determined by the cellular stage of differentiation. The identification of these genes along with the analysis of the role played by them will provide a better understanding of the role played by the ubiquitin proteasome pathway during ETD.